Cytoplasmic male sterility system producing canola hybrids

ABSTRACT

Our invention comprises a gene restorer line of  Brassica napus  which contains a  Raphanus sativus  restorer gene but is essentially free of  Raphanus sativus  genes which produce high glucosinolate. In particular, we provide a gene restorer line, and progeny derived therefrom, seed of which is low in glucosinolates. The  Brassica napus  restorer lines are free of glucosinolate-producing genes having a characteristic RFLP signature. The method of producing such lines which comprises crossing  Brassica napus  restorer lines and hybrids with desired  Brassica napus  germplasm and selecting progeny having a characteristic RFLP signature is also encompassed by the present invention.

[0001] This application is entitled to the benefits of foreign priorityunder Title 35 U.S.C. section 119. The foreign priority document isUnited Kingdom application 9513881.4 filed on 07 Jul. 1995.

FIELD OF THE INVENTION

[0002] This invention relates to improved plants. In particular, itrelates to new plant germplasm of the Brassica species, having a reducedcontent of undesired glucosinolates.

BACKGROUND OF THE INVENTION

[0003] Economic production of Brassica spp. hybrids requires apollination control system and effective transfer of pollen from oneparent to the other. The ogura cytoplasmic male sterility (cms) system,developed via protoplast fusion between radish (Raphanus sativus) andrapeseed (Brassica napus) is one of the most promising methods of hybridproduction. It provides stable expression of the male sterility trait(Ogura 1968), Pelletier et al. (1983) and an effective nuclear restorergene (Heyn 1976).

[0004] Initial restorer material showed reduced female fertility whichwas overcome through backcrossing. Delourme et al. (1991) attributedthis to elimination of a portion of the radish chromosome that had beenintroduced along with the restorer gene. In their work, successivebackcross generations produced fertility levels successively closer tonormal.

[0005] High glucosinolate (GSL) content in seed of Brassica napus is ananti-nutritional factor. Meal made from such seed is unsuitable for usein animal feeds. Seed GSL level is an expression of the genotype of thefemale plant and is determined by four to eight separate dominant andadditive genes. Two to five genes are involved in alkenyl (one of thealiphatic group) glucosinolate content, while two or three genes areinvolved in indole glucosinolate content (Rücker and Röbbelen, 1994).Total aliphatics may be determined by up to six genes (Magrath et al.1993).

SUMMARY OF THE INVENTION

[0006] An object of the present invention is to provide Brassica spp.hybrids, seeds, microspores, ovules, pollen, vegetative parts containinglow glucosinolate and the restorer gene.

[0007] Yet another object of the present invention is to provideinterspecific crosses using fertile, low glucosinolate plants with theogura cytoplasm as the female, followed by selection for fertility andlow glucosinolate.

[0008] A further object of the invention is to provide a method foridentifying a restorer line that contains only the portion of theRaphanus sativus material necessary for fertility and not the portion ofthe Raphanus sativus material that produces high glucosinolate.

[0009] Accordingly, our invention comprises a gene restorer line ofBrassica napus which contains a Raphanus sativus restorer gene but isessentially free of Raphanus sativus glucosinolate-producing genes. Inparticular, we provide the gene restorer line KH, and progeny derivedtherefrom, seed of which is low in glucosinolates. We further provideBrassica napus restorer lines free of glucosinolate-producing geneshaving a characteristic RFLP signature, as hereinafter described, and amethod of producing such lines which comprises crossing Brassica napusrestorer and/or hybrid lines with desired Brassica napus germplasm andselecting progeny having a characteristic RFLP signature. Clearly thisinvention encompasses hybrids containing the restorer gene without thehigh glucosinolate material. Additionally, these hybrids can be used tocreate new restorer lines within the scope of this invention.

[0010] The present invention broadly includes a method of producing animproved restorer line of Brassica for use in a cytoplasmic malesterility system, which comprises forming a plant population from a generestorer line of Brassica napus which contains a Raphanus sativusrestorer gene and Raphanus sativus glucosinolate genes. Then breedingwith the progeny of the plant population. Furthermore, it includestesting the progeny for fertility indicating the Raphanus sativusrestorer gene is present and for levels of glucosinolate wherein thepresence and absence of Raphanus sativus high glucosinolate productionis shown; and selecting progeny which are positive for presence of therestorer gene and negative for the Raphanus sativus with glucosinolateproduction.

[0011] The inventive methods of this application also include a methodof forming Brassica napus hybrid seed and progeny thereof from acytoplasmic male sterility system which includes a restorer linecontaining Raphanus sativus restorer gene. This method includes thesteps of providing a homozygous improved restorer line produced, asoutlined above, using the restorer line in a hybrid production field asthe pollinator; using cytoplasmic male sterile plants in a hybridproduction field as the hybrid seed producing plant; and harvesting thehybrid seed from the male sterile plant.

[0012] Additionally, when producing progeny, the method includes thestep of planting the hybrid seed from the male sterile plant and growinga plant therefrom.

[0013] The present invention clearly shows how to form an improvedBrassica ssp., an improved Brassica napus plant, having lowglucosinolate seeds, the plant containing Raphanus sativus gene materialthat is capable of restoring fertility to the ogura cytoplasmic malesterile plants, the improvement comprising an improved Brassica napusplant evidencing deficient glucosinolate production from the Raphanussativus material, wherein the improved plant produced low glucosinolateseeds.

[0014] A Brassica napus plant containing Raphanus sativus restorer geneunlinked from Raphanus sativus glucosinolate genes adapted to restorefertility to ogura cytoplasmic male sterile.

[0015] The present invention describes the molecular marker method. Thisis a method wherein the markers mapping to similar regions as those inthe group consisting of, WG3F7, TG1H12, OPC2, WG4D10, WG6F3 are employedto identify the Raphanus sativus material which contains highglucosinolate producing genes.

[0016] The present invention encompasses not only canola ouality but anylow glucosinolate material produced for a cytoplasmic sterile plantcontaining Raphanus sativus. Any canola quality (erucic acid<2% and <30umoles glucosinolates/gram defatted dry meal) restorer line, capable ofinducing fertility in Brassica plants containing the INRA Oguracytoplasmic male sterility. Further, the present invention encompassesBrassica spp. hybrids, seeds, microspores, ovules, pollen, vegetativeparts containing low glucosinolate restorer gene. Interspecific crossesusing fertile, low glucosinolate plants with the ogura cytoplasm as thefemale, followed by selection for fertility and low glucosinolate.

[0017]Brassica spp. hybrids, seeds, microspores, ovules, pollen,vegetative parts containing low glucosinolate restorer gene asidentified by using probes such as those as described herein.

[0018] Additionally in the broad scope of the invention included is theBrassica napus (spring and winter types) or B. rapa containing the lowglucosinolate restorer gene as described.

BRIEF DESCRIPTION OF THE DRAWING

[0019]FIG. 1 is a schematic map showing the relation of high GSL genesto the restorer gene in ogura germplasm, as revealed by our work, andthe location of probes binding in this area.

DETAILED DESCRIPTION OF INVENTION

[0020] We now describe genes for high seed glucosinolate content (GSL)which were also introduced with the restorer gene. In addition, wedescribe our work which has broken the very tight linkage between theradish-derived restorer gene and the non-canola quality levels ofglucosinolates in the seed. The resulting lines are the first canolaquality restorers for this cms system, which in turn produce the firstfully fertile ogura cms canola hybrids. The terms hybrid, line and plantor progeny when used in the claims includes but are not limited toseeds, microspores, protoplasts, cells, ovulas, pollen, vegetativeparts, cotyledons, zygotes and the like.

[0021] Background

[0022] The original Brassica napus restorer material, RF, used in ourwork, is an F6 line from the cross

FU58.Darmor BC1/Rest.Darmor BC1//Bienvenu,

[0023] and was obtained from the Institut National de RecherchesAgricoles (INRA) in 1992. This material is commercially available underlicense from INRA. This material is biennial, low erucic acid (C22:1)and high GSL. It therefore required backcrossing into elite spring typesfor use in our spring hybrid program.

[0024] All fertile F1 plants from RF crossed by spring lines tested highfor aliphatic glucosinolate as expected. However, corresponding sterilespossessed GSL levels of less than 30 μmoles/gram defatted dry meal. Thisindicated an extremely tight linkage between the restorer and high GSLgenes. Absence of high GSL sterile plants also indicated the lack ofhigh GSL genes normally found in rapeseed. Except for the presence ofthe radish GSL genes, fertile plants should therefore have been canolaquality. High GSL content in seed of fertile plants therefore wasderived from radish DNA inserted with the restorer gene.

[0025] Based on the theory of a single dominant gene for fertilityrestoration and another single dominant gene complex for GSL content,individual plants were expected to segregate as follows in subsequentbackcross generations:

[0026] ½ male sterile

[0027] ¼ high GSL, fertile

[0028] ¼ low GSL, fertile

[0029] Of 493 BC1 crosses studied, no low GSL fertile plants wereobtained. Over 298 BC2 crosses also failed to produce low GSL restorers.This again points to a very strong linkage between the restorer gene andthe radish-derived high GSL genes. Restored plants possessed elevatedlevels of progoitrin and gluconapin compared to control plants. Levelsof sinapine, glucoalysin and glucobrassicanapin fluctuated in therestored plants relative to controls (Table 1).

[0030] Delourme et al. (1994), using RAPD markers, concluded that radishDNA had been retained around the restorer gene. Our RFLP data showedthat the portion of the Raphanus chromosome which was introgressed intothe Brassica genome contained the radish high GSL genes in addition tothe restorer.

[0031] The absence of low GSL restorers was observed as far as the BC7generation in the 1994 Zeneca Seeds nursery in Carman, Manitoba. Over700 backcrosses (BC1 to BC6) were performed in the 1994 field programusing emasculated fertile plants containing the ogura cytoplasm(therefore containing the restorer gene) as the female. In addition,over 500 doubled haploids from various restorer by germplasm crosseswere evaluated. All doubled haploids were high (over 30 μmoles/gram)GSL.

[0032] Of the 700 backcrosses, three gave rise to seed which was foundto have low (<30 μmoles/gram dry seed) GSL levels, equal to sterileplants in the row. All three (KH-A, KH-B, KH-C) were BC2 progeny of theproprietary Zeneca Seeds line BNO559 originally crossed to a restorergene source KH in November 1993. The restorer gene source KH for theline was a BC1 plant of the original restorer source from INRA (RF)crossed twice to a Zeneca Seeds inbred 4372 (RF<2<4372). Review of thehistory of the line KH (RF<2<4372)<2<BNO559 indicated a prior generationof low GSL results in the controlled environment growth room. TABLE 1Comparison of Glucosinolate Profiles of High GSL Restored Plants inogura Cytoplasm and Corresponding Fertile Parent in Normal Cytoplasm*. #Pedigree PRO EPI SIN NAP ALY GNA 40H GBN GBC NAS NEO ALI IND TOT 1RF<5<BN0027-22-1-1 11.31 0.36 1.26 0.18 0.19 3.13 2.56 0.22 0.21 0.290.02 16.64 3.08 19.72 2 RF<5<BN0027-22-1-2 32.13 0.84 6.42 0.44 1.737.75 3.09 0.72 0.19 0.91 0.06 49.83 4.26 54.09 7 RF<4<BN0027-22-1-230.89 0.60 7.23 0.26 0.94 8.94 2.09 0.30 0.42 2.96 0.01 49.16 5.49 54.648 RF<S<BN0027-22-1-2 29.77 0.58 5.08 0.28 0.93 7.58 1.96 0.48 0.50 1.820.01 44.70 4.29 48.99 13 RF<5<BN0027-22-1-3 22.74 0.45 2.80 0.16 1.168.26 2.04 0.88 0.27 2.33 0.02 36.45 4.65 41.10 14 RF<5<BN0027-22-1-322.37 0.44 3.19 0.18 0.61 5.01 1.77 0.23 0.35 1.53 0.02 32.04 3.87 35.7215 BN0027-22-1-1 2.27 0.04 1.47 0.12 0.06 0.60 1.52 0.23 0.32 1.64 0.034.80 3.51 8.31 16 BN0027-22-1-2 1.79 0.04 1.28 0.11 0.05 0.49 1.50 0.020.16 1.97 0.05 3.78 3.68 7.47 17 BN0027-22-1-3 1.12 0.02 0.00 0.10 0.030.34 1.20 0.20 0.09 0.98 0.02 1.82 2.29 4.10 Legend for Table 1ALIPHATIC INDOLE GLUCOSINOLATE Code GLUCOSINOLATE Code MISC. CodeProgoitrin PRO 4-Hydroxy 4OH Total ALI Glucobrassicin aliphaticsEpiprogoitrin EPI Glucobrassicin GBC Total indoles IND Sinigrin SINGluconasturtiin NAS Total GSL TOT Napolederin NAP Neoglucobrassicin NEOGlucoalysin ALY Gluconapin GNA Glucobrassicanapin GBN

[0033] To verify that KH was in fact a low GSL restorer (R) line, athree-step approach was used.

[0034] 1) GSL levels of subsequent generations were again evaluated inthe field,

[0035] 2) Genetic studies were conducted to verify inheritance of therestorer gene-and

[0036] 3) RFLP analyses were used to determine differences between highGSL and canola-quality lines or low GSL lines and plants.

[0037] 1) Verification of Glucosinolate Levels

[0038] Material was grown in the nursery (November 94-March 95) inTasmania, Australia for glucosinolate evaluation of a third generation.The three low GSL BC2 lines KH-A, -B, -C, were planted in three separaterows, along with high GSL sister lines (different original cross toBNO559) and non-related restorers in adjacent plots. Since expression ofGSL content in the seed is not affected by pollen source (Magrath et el.1993), both selfed and open-pollinated seed was tested from these rows.As shown in Table 3, only plants descended from KH, the originalRF<3<BNO559-3-2, were again low GSL. Sister lines also derived fromBNO559 were not. Thus it appears that the break in the linkage betweenthe restorer gene and the adjacent high GSL genes occurred as the resultof a specific meiotic event which was “captured” in one cross (Table 2).All radish-derived GSL genes were lost in the one event; therefore, theyhad been tightly linked together as a complex acting like a singledominant gene linked to the restorer gene. TABLE 2 Source Gluc (9)*RF<3<(BNO559)-1-2-1)-1 5.4 RF<3<(BN0559)-1-2-1)-2 4.5RF<3<(BNO559)-2-2-2)-1 5.5 RF<3<(BNO559)-2-2-2)-2 6.6RF<3<(BNO559)-2-4-1)-1 4.4 RF<3<(BNO559)-2-4-1)-2 5.4RF<3<(BNO559)-3-1-1)-1 4.4 RF<3<(BNO559)-3-1-1)-2 4.4RF<3<(BNO559)-3-2-1)-1 2.2 RF<3<(BN0559)-3-2-1)-2 3.2RF<3<(BN0559)-3-2-2)-1 2.2 RF<3<(BN0559)-3-2-2)-2 2.2RF<3<(BN0559)-3-2-2)-3 2.3 RF<3<(BN0559)-3-2-3)-1 3.2RF<3<(BN0559)-3-2-3)-2 2.3 RF<3<(BN0559)-4-3-2)-1 4.5RF<3<(BN0559)-4-3-2)-2 3.4

[0039] Table 2—shows glucosinolate results from Tasmania nursery1994-95. Bolded cells indicate progeny of low GSL row in 1994 Carmannursery. (GSL ratings 1-9 using Tes-Tape method, where canola quality<3. *Duplicate analyses performed on each sample).

[0040] There are at least two well known methods of testing forglucosinolate. The first test is for quantitative glucosinolate analysisusing high performance liquid chromatography. This test is cited in ISOMethod 9167-1:1992. Rapeseed—Determination of glucosinolatescontent—Part 1: Method using high-performance liquid chromatography,International Organization for Standardization, Geneva.

[0041] The second test is described below:

[0042] The Tes-Tape Method for Evaluation of Seed Glucosinolate Contentin Brassicas. (Based on Rakow et al. (1981).

[0043] 1. Place 5 seeds in a microtitre plate well.

[0044] 2. Crush seed using a rod and light hammer stroke, cleaning rodbetween samples.

[0045] 3. Add 100 μL (microlitres) of distilled water or 100 μL or 1millimolar sodium ascorbate if seed is old (reduced viability).

[0046] 4. Wait 10 minutes.

[0047] 5. Add 25 μL of 70 g/L charcoal solution.

[0048] 6. Wait 1 minutes.

[0049] 7. Insert a 2 cm strip of Tes-Tape (normally used to test forglucose content in urine of diabetics).

[0050] 8. Wait 5 minutes.

[0051] 9. Read Tes-Tape color change. Color change may be based oneither a 1-5 or 1-9 scale as follows:

[0052] The low GSL trait was expressed for a third consecutivegeneration in progeny of the RF<2<BNO559-3-2 line (bolded rows). Allplants harvested from the line were canola-quality. Sister lines andnon-related strains (data not shown) were all high (rapeseed levels).Using a Wilcoxon Rank Test, with normal approximation and a continuitycorrection of 0.5, the GSL values of the identified line weresignificantly lower than closely related sister lines (p=0.0001).Statistically, this line is significantly lower in glucosinolates thanany other ogura restorer.

[0053] 2) Verification of Restorer Gene using Genetic Studies

[0054] 2.a) Testcrosses

[0055] The putative restorer line KH, RF<3<BNO559-3-2, was crossed tofive genetically-diverse male-sterile lines possessing the oguracytoplasm. Since the restorer gene was first identified in abackcross-derived line, F1 plants derived from these crosses wereexpected to segregate evenly for fertiles and steriles. As shown inTables 3 and 4A, testcross progeny data support the concept of a singledominant gene for restoration. TABLE 3 Female # Steriles # Fertiles 1 3634 2 50 43 3 78 63 4 71 67 5 76 73 Observed Total 311 280 Expected Total295.5 295.5

[0056] Table 3—testcross results using BC2F1 plants as restorer genesource.

[0057] The Chi-Square value calculated for Goodness of Fit of theseresults to the expected 1:1 ratio is 1.626 with 1 degree of freedom(p=0.20). The results are therefore not statistically distinguishablefrom those expected (Steele and Torrie, 1980).

[0058] 2.b) F2 Segregation Ratios

[0059] BC2 plants were also selfed in order to determine segregationratios of the BC2F2 population. Six hundred and eighty-six single F2plants were evaluated for fertility status. Based on the assumption of asingle dominant gene originally introduced from the radish parent, theF2 population should have segregated 3 Fertile: 1 Sterile. As shown inTable 5, observed results were close to expected values. TABLE 4A ClassFertile Sterile Number of plants observed 499 187 Theoretical number ofplants expected 514.5 171.5

[0060] Table 4A—Frequency distribution of F2 population.

[0061] The Chi-Square value for Goodness of Fit calculated for theseresults is 1.868 with 1 degree of freedom (p=0.17). The results aretherefore not statistically different from expected values (Steele andTorrie, 1980).

[0062] Examples of using hybrid as source of restorer gene

[0063] Selfing Down of Hybrid

[0064] Low glucosinolate hybrids containing the new restorer gene weregrown out. Fertile plants were self pollinated, some with bags, othersby brushing pollen manually. F2 seed was harvested from these F1 plantsand planted as a population. Fertile plants from the population wereselected and grown as F3 rows, thereby providing starting material forbreeding approaches such as pedigree breeding, recurrent selection andothers.

[0065] As Parent in Traditional Breeding

[0066] Lines containing the improved restorer gene were crossed withother germplasm lines as part of the breeding program. The F1 from thesecrosses was grown out. Fertile plants were self pollinated and resultantF2 seed harvested. Fertile plants from the F2 population were selected,harvested and grown as F3 rows, thereby providing starting material forbreeding approaches such as pedigree breeding, recurrent selection andothers.

[0067] As Parent in Doubled Haploid

[0068] A source of the improved restorer gene was crossed to improvedgermplasm. The resulting hybrids, 94-0186 and 94-0187, underwentmicrospore culture to produce doubled haploid restorer lines. Microsporeculture methods utilized were similar to those described by Chen et al(1994) and Mollers et al (1994). These restorer lines have been verifiedas low glucosinolate.

[0069] As a Source of Restorer in Backcross Program

[0070] Material containing the improved restorer gene was crossed toselected Zeneca Seeds' inbred lines. Fertile plants were emasculated andcrossed again to the inbred line (recurrent parent). Resulting fertileswere backcrossed again to the inbred line. At any generation, selfingdown of material could begin to produce new restorer lines. Theseprojects exemplify a backcrossing program to bring the restorer geneinto superior germplasm. The RFLP analysis could be employed to assistin early selection of plants having a favorably marker signature for lowglucosinolate production in combination with having the restorer gene.

[0071] Field Segregation

[0072] F3 rows from BN0611 were planted in the nursery. The expectedsegregation ratio was 2:1 (segregating rows: fully fertile rows). Somerows exhibited very poor emergence with most of these containing onlyfertile plants. Unexpectedly, the segregation results were 340segregating to 105 fertile, far from the 2:1 ratio expected from asingle gene inheritance.

[0073] Doubled Haploids

[0074] The original BN0611 (a BC2 line) underwent microspore culture toproduce true-breeding restorer lines. Again, unexpectedly, of the plantswhich successfully underwent chromosome doubling, the proportion offertiles was vastly less than expected. The frequency was 254 steriles:106 fertiles instead of a 1:1 ratio. These results, combined with fieldresults, may indicate that low glucosinolate restoration is controlledby more than a single dominant gene or that the Raphanus sativusmaterial is not well integrated into the genome. Additional theories mayultimately give other reasons for this unexpected segregation ratio.

[0075] Testcrosses

[0076] Twenty BN0611 F3 rows were chosen for being homozygous for therestorer gene. A single plant from each row was crossed to a malesterile line. F1 seeds were planted from each testcross and allowed toflower, at which time fertility of the F1 plants were evaluated. CrossMale Steriles Fertiles Haploids 0089 BN0611-1)-2-2}:11 0 6 0 0090BN0611-1)-2-4}:11 0 24 0 0091 BN0611-1)-3-4}:11 0 19 0 0092BN0611-1)-8-2}:11 0 13 0 0093 BN0611-1)-10-3}:11 0 27 0 0094BN0611-1)-16-2} 0 24 0 0095 BN0611-1)-22-1}:11 0 26 1 0096BN0611-1)-22-3}:11 0 15 1 0097 BN0611-1)-22-4} 0 24 0 0098BN0611-1)-22-5}:11 0 26 0 0099 BN0611-1)-28-3}:11 0 22 1 0100BN0611-1)-31-1}:11 0 6 1 0101 BN0611-1)-31-4}:11 0 17 1 0102BN0611-2)-7-2}:11 0 25 2 0103 BN0611-2)-7-3}:11 14 9 1 0104BN0611-2)-7-6}:11 15 11 0 0105 BN0611-2)-8-5}:11 0 5 0 0106BN0611-2)-9-5}:11 0 21 1 0107 BN0611-2)-11-4}:11 0 21 0 0108BN0611-2)-11-5}:11 0 26 0

[0077] The fertile plants did exhibit some abnormal characteristics suchas missing petals, malformed buds and bent stigmas. The severity ofthese traits varied by cross, suggesting some genetic influence by themale.

[0078] Crosses 103 and 104 shows a 1:1 segregation. Emergence data fromthe field showed that these two males had very few plants in the row,and thus had been mis-classified “homozygous”.

[0079] Many F3 lines included a plant which had traits associated withhaploids, i.e. very small buds and flowers. These plants also appearedto have a different leaf type than the other F1's, having a deeper lobedleaf. It may be possible that these plants are aneuploids, and that theextra genetic material could be causing the observed difference in leafmorphology.

[0080] New F3 Lines

[0081] The three low glucosinolate lines crossed by B line have beentested for segregation ratio of the F2 and F3 plants. Table 4B showsresults again distinctly different from expected ratios. TABLE 4B CrossF2 Fertile F2 Sterile F3 Segregating F3 Fertile 0181 n/a n/a 140(110)25(55) 0184 67(81) 41(27) 57(43) 07(21) 0189 119(118) 38(39) 146(116)28(58)

[0082] These results are far from the expected ratio of two segregatingF3 lines for every homozygous line. There is frequently a bias towardfewer fertiles than would be expected from a single gene as the geneapproaches homozygosity.

[0083] Glucosinolate Data

[0084] Quantitative glucosinolate data on a number of the lines areincluded in the following Table 4C. TABLE 4C Pedigree PRO EPI SIN NAPALY GNA 4OH GBN GBC NAS NEO ALI IND TOT RF<5<BN0027-22-1-1 11.31 0.361.26 0.18 0.19 3.13 2.56 0.22 0.21 0.29 0.02 16.64 3.08 19.72RF<5<BN0027-22-1-2 32.13 0.64 6.42 0.44 1.73 7.75 3.09 0.72 0.19 0.910.06 49.83 4.26 54.09 RF<4<BN0027-22-1-2 30.89 0.60 7.23 0.26 0.94 8.942.09 0.30 0.42 2.96 0.01 49.16 5.49 54.64 RF<5<BN0027-22-1-2 29.77 0.585.08 0.28 0.93 7.58 1.96 0.48 0.50 1.82 0.01 44.70 4.29 48.99RF<5<BN0027-22-1-3 22.74 0.45 2.80 0.16 1.16 8.28 2.04 0.88 0.27 2.330.02 36.45 4.65 41.10 RF<5<BN0027-22-1-3 22.37 0.44 3.19 0.18 0.61 5.011.77 0.23 0.35 1.53 0.02 32.04 3.87 35.72 BN0027-22-1-1 2.27 0.04 1.470.12 0.06 0.60 1.52 0.23 0.32 1.64 0.03 4.80 3.51 8.31 BN0027-22-1-21.79 0.04 1.28 0.11 0.05 0.49 1.50 0.02 0.16 1.97 0.05 3.78 3.88 7.47BN0027-22-1-3 1.12 0.20 0.00 0.10 0.03 0.34 1.20 0.20 0.09 0.98 0.021.82 2.29 4.10 RF<3<(BN0559)-3-2-1)-8-2 4.39 0.01 0.12 1.70 6.77 2.790.01 0.32 0.02 9.79 7.19 16.98 RF<3<(BN0559)-3-2-2)-16-5 4.82 0.01 0.111.41 3.91 2.41 0.10 0.24 0.02 9.45 4.32 13.77 RF<3<(BN0559)-3-2-3)-27-22.85 0.01 0.08 1.54 4.07 1.26 0.08 0.08 0.01 6.12 4.37 10.49 BN0111 +BN0018 check 1.85 0.01 0.06 0.95 4.15 2.40 0.09 0.08 0.03 5.62 4.5010.12

[0085] The second group of data (on the previous page) comes from theCarman, Manitoba breeding nursery. As expected, there are some changesin levels of individual glucosinolates due to environmental factors(Mailer and Cornish, 1987). However, it is clear that the level ofprogoitrin (2-Hydroxy-4-pentenylglucosinolate) and gluconapine aresignificantly lower in the RF<3<(BN0559)-3-2 derived lines than in highglucosinolate material with the original restorer gene obtained fromINRA.

[0086] 3) RFLP Results

[0087] 3.a) Mapping of the Restorer Gene Locus

[0088] In order to determine the position of the restorer gene on theBrassica napus genetic map, DNA was purified from members of a BC1population that was segregating for the presence of the restorer gene(scored as male fertility in a sterile cytoplasm). The DNA samples weredigested with restriction endonucleases, subjected to agarose gelelectrophoresis, and transferred to nylon membranes (essentially asdescribed by Southern, 1975). The membranes were then treated withheat-denatured, ³²P-labeled DNA probes (Sharpe et al, Osborn et al) and,following overnight hybridization and washing at an appropriatestringency, subjected to autoradiography. The RFLP patterns revealed bythese probes were noted, and the probes giving bands of hybridizationshowing linkage to the restorer phenotype are shown in Table 5. A numberof characteristic (“diagnostic”) alleles were seen at the RFLP locilinked to the restorer locus, that are not present in the majority ofcanola germplasm. In addition to the RFLP probes, one oligonucleotideprimer was used to generate RAPD patterns, recently published as beinglinked to the Restorer gene (Landry et al., 1994); this is also shown inTable 5. The use of AFLP, RFLP, RAPD, microsatellites, primer and otherprobes, etc. to give genetic fingerprints of the Raphanus sativusmaterial and surrounding Brassica material is encompassed within thescope of this invention.

[0089] 3.b) Characterization of Low GSL Fertile Recombinants

[0090] Representative samples from the backcrosses that generated lowGSL recombinants, described in sections 1 and 2, above, were analyzedwith the probes listed in Table 5. The tight linkage between therestorer gene and the diagnostic RFLP alleles was maintained in the widerange of crosses being studied. Two recombination events are shown. Thediagnostic alleles “lost” in these plants permit their loci to be placedin a slightly random order along the chromosome, relative to therestorer locus (illustrated in FIG. 1). Two separate recombinationevents have occurred—one in family BNO599, in which the high GSL regionhas been separated from the restorer, and a second in family 4504, wherethe restorer region has also been lost.

[0091] The GSL levels of the various plants are shown alongside thegenotypes in Table 6. (GLS levels were -measured by the HPLC method forevaluation of seed glucosinolate content in Brassicas. This indicatesthat the gene encoding high GSL levels is linked to the diagnosticalleles, and lies on the segment of chromosome marked by pO120, pO119and pN64. Because of the low frequency of recombination in this regionof the genome, it is impossible to quote precise distances. However, itis clear that by selecting fertile plants that lack the diagnosticalleles for the linked loci, it should be possible to improve thefrequency of low GSL fertile plants in the backcross progeny. TABLE 6RFLP/RAPD locus Gluco- GSL Segregating OPC2 Fer- sino- (2 plant pN213AWG3F7D TG1H12D 1150 WG4D10J pO9O tility pN64D pO119H pO120F WG6F3E latesreps) FR2<2<4504- + + + + + + F + + + + H 67.5, 1-1/4504-1-1 68.1 pl 6FR2<2<4504- − − − − ? − S − − − − L  8.3, 1-1/4504-1-1 10.9 pl 12FR2<2<4504- + + + + + + F + + + + H 69.9, 1-1/4504-1-1 69.6 pl 5FR2<2<4504- + + + + + + F + + + + H 67.5, 1-1/4504-1-1 68.01 pl 6FR2<2<4504- + + − − ? − S − − − − L  8.2, 1-1/4504-1-1  7.2 pl 17RF930307<3< + + + + + + F − − − − L 15.4, BN0559-3-2-2 17.7 pl 3RF930307<3< + + + + + + F − − − − L 28.2, BN0559-3-2-2 28.5 pl 10RF930307<3< − − − − − − S − − − − L 26.9, BN0559-3-2-2 27.6 pl 7RF930307<3< − − − − − − S − − − − L 26.8, BN0559-3-2-2 26.7 pl 4

[0092] TABLE 7 Allele Origin associated Probe (see with Approx .allelename note 1) Enzyme Restorer size (bp)* pN213 1 EcoRI A 23000 WG3F7 3EcoRI D 7000 TG1H12 3 EcoRI D 3700 OPC2 4 1150 1150 WG4D10 3 EcoRI J3400 pO9 2 EcoRI O 19000 pN64 1 EcoRI D 4300 pO119 2 EcoRI H 6500 pO1202 EcoRI F 4600 WG6F3 3 EcoRI E 13000

[0093] Not only has the present invention been implemented in Brassicanapus but it also had been implemented in other Brassica spp.

[0094] Rapa Work with this Gene

[0095] The low glucosinolate gene has been backcrossed into Zeneca B.rapa lines as follows:

[0096] At the BC2 generation, both fertile and sterile plants have beenobtained in an approximately 50:50 ratio. The plants are morphologicallyidentical to the recurrent B. rapa parent. It is apparent that therestorer gene has been successfully introduced into the Brassica rapaspecies. Similar crossing techniques could be utilized to introduce thisrestorer gene into other Brassica species as well.

[0097] Conclusion

[0098] We have produced a clear improvement in the INRA ogura cms systemof producing hybrid canolas. A strong linkage between the restorer geneintroduced from Raphanus sativus and high glucosinolate genes from thesame source was broken through an intensive crossing program. Based onthe literature and all other publicly available information, there wereno lines available to produce low glucosinolate, restored hybrids usingthe ogura cytoplasm until this work. It will now be possible to use thismaterial (KH, and lines derived from it) as a source of fertility in allfuture canola-quality fertile Brassica hybrids using the oguracytoplasm.

[0099] Furthermore, using the information given herein about where theprobes used are located on the genome of ogura germplasm, it will bepossible to use probes to test germplasm of this type to determine if ithas the desired combination of restorer gene and low GLS. Accordingly,it is a further feature of our invention to provide ogura germplasmwhich gives a signal with probes binding in the restorer gene region ofthe genome, as shown in FIG. 1, but no signal with probes binding in thehigh GSL region of FIG. 1.

[0100] References

[0101] Chen, Z. Z., S. Snyder, Z. G. Fan and W. H. Loh 1994. Efficientproduction of doubled haploid plants through chromosome doubling ofisolated microspores in Brassica napus. Plant Breeding 113:217-221.

[0102] Delourme, R., F. Eber and M. Renard. 1991. Radish cytoplasmicmale sterility in rapeseed: breeding restorer lines with a good femalefertility. Proc 8th Int Rapeseed Conf. Saskatoon, Canada. pp. 1506-1510.

[0103] Delourme, R., A. Bouchereau, N. Hubert, M. Renard and B. S.Landry. 1994. Identification of RAPD markers linked to a fertilityrestorer gene for the Ogura radish cytoplasmic male sterility ofrapeseed (Brassica napus L.). Theor Appl Genet. 88:741-748.

[0104] Heyn, F. W. 1976. Transfer of restorer genes from Raphanus tocytoplasmic male-sterile Brassica napus. Cruciferae Newsletter. 1:15-16.

[0105] Magrath, R.,: C. Herron, A. Giamoustaris and R. Mithen. 1993. Theinheritance of aliphatic glucosinolates in Brassica maps. Plant Breeding111: 55-72.

[0106] Ogura, H. 1968. Studies on the new male sterility in Japaneseradish, with special reference on the utilization of this sterilitytowards the practical raising of hybrid seeds. Mem Fac Agric KagoshimaUniv. 6: 39-78.

[0107] Pelletier, G., C. Primard, F. Vedel, P. Chétrit, R. Rémy, P.Rousselle and M. Renard. 1983. Intergeneric cytoplasmic hybridization inCruciferae by protoplast fusion. Mol Gen Genet. 191: 244-250.

[0108] Rakow, D., R. Gmelin and W. Thies. 1981. Enzymatische Darstellungund Eigenschaften einiger Desulfoglucosinolate. Z Naturforsch. 36:16-22.

[0109] Mailer, R. J. and P. S. Cornish. 1987. Effects of water stress onglucosinolate and oil concentrations in the seed of rape (Brassica napusl.) and turnip rape (Brassica rapa L. var. silvestris±Lam.Fr Briggs).Aust. J. Exp. Agric. 27:707-711.

[0110] Mollers, C., M. C. M. Iqbal and G. Robbelen. 1994. Efficientproduction of doubled haploid Brassica napus plants by colchicinetreatment of microspores. Euphytica 75:95-104.

[0111] R{overscore (u)}cker, B. and G. R{overscore (u)}bbelen. 1994.Inheritance of total and individual glucosinolate contents in seeds ofwinter oilseed rape (Brassica napus L.). Plant Breeding. 113: 206-216.

[0112] Steele, R. G. D. and J. H. Torrie. 1980. Principles andProcedures of Statistics. McGraw-Hill Book Company.

We claim:
 1. A method of producing a restorer line of Brassica havingsubstantially the same glucosinolate level as a corresponding fertileparent for use in an ogura cytoplasmic male sterility system comprising:A. selecting a fertile parent with microspores comprising a generestorer line of Brassica napus which contains a Raphanus sativusrestorer gene and canola quality levels of glucosinolate particularlylevels of progoitrin and gluconasin glucosinolate which are canolalevels; B. culturing selected microspores forming haploids and inducingdouble haploids; C. testing the double haploids progeny for fertilityindicating the Raphanus sativus restorer gene is present and for levelsof glucosinolate wherein the absence of levels of progoitrin andgluconasin glucosinolate and overall glucosinolate production is shownto be substantially the same as the corresponding fertile parent; and D.selecting progeny which are positive for presence of said restorer geneand negative for elevated glucosinolate production relative to thecorresponding fertile parent.